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Cell Culture Facility |
The Protein Microanalytical Laboratory (previously known as the Protein Sequencing Facility) is a non-profit center situated in 301 Clapp Hall in the Department of Biological Sciences (consult our building floor plan for precise location within Clapp Hall).. The Laboratory provides investigators with amino acid sequence and composition data, including chemical (Edman) sequencing of peptides and proteins. In recent years, the majority of samples for N-terminal sequencing have been submitted as Coomassie-stained blots on PVDF membranes from acrylamide gels. While this technique is widespread, and can rapidly yield results, it is frequently not appreciated that, by some estimated, 80% of all proteins are N-terminally modified, rendering them refractive to Edman degradation. Most often, the modification is N-acetylation, which is nearly impossible to selectively remove. To further complicate matters, proteins with free amino groups may be acylated by unpolymerized acrylamide encountered during electrophoretic separation. To overcome these difficulties, this laboratory has had great success with "in-gel" digestion of a variety of gel-banded proteins from different investigators. Unless identification of the N-terminal sequence of the intact protein is specifically required, in-gel digestion appears to be the perfect solution to the above problems. While techniques are also available to digest proteins from PVDF blots, in-gel digestion circumvents all trans-blotting and the accompanying losses. The recovery of peptides from the digest appears to be nearly quantitative. The proteins are digested with a protease, endoLys-C, generating a mixture of peptides that can be subsequently separated by High Performance Liquid Chromatography. On the average, at least 10 residues of sequence can be obtained from each peptide. To avoid modifying the protein during electrophoresis, age the gels before using them. Another tip that may be valuable is to flush the gel well with buffer before loading the sample. Once these precautions have been taken even dried gels or gels stored in notebooks can be successfully used to sequencing. John Hempel and the protein sequencer Fee Schedule
Protein Microanalytical Laboratory
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